Plasmids play a central role in the spread of resistance among bacterial species thereby decreasing the effectiveness of various chemotherapeutic agents for the treatment of infectious diseases. They carry genes that encode essential functions, such as replication, maintenance and transfer, as well as a variety of accessory functions, such as antibiotic resistance determinants. The objective of the proposed research is to obtain a more systematic and comprehensive understanding of plasmid evolution through experimental evolution studies. The specific aims are 1) To assess the tempo and mechanisms of plasmid evolution during vertical transmission in a single host as compared to vertical and horizontal transmission among phylogenetically distinct hosts, in the presence of selective pressure; 2) To characterize and compare the genetic and phenotypic changes that occur during such experimental plasmid evolution; 3) To test the ability of various algorithms to accurately reconstruct the true phylogenies of independently evolved plasmids and specific genes. The broad host range Inc-1beta plasmid pB10, which encodes resistance to four antibiotics and mercury, will be experimentally evolved in replicate cultures of three genetically distinct hosts (Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia) as previously described in studies of microbial evolution. Plasmid evolution in one single host will be compared with evolution in alternating hosts, and in each case plasmids evolved for differing periods of time will be characterized. Phenotypic changes will be characterized by examining the effect of the evolved plasmid on host fitness and by assessing differences in the stability and broad host range characteristics of the evolved and ancestral plasmids. Genetic changes that may account for the observed phenotypic differences will be identified by characterizing macroscale and microscale variations in the evolved replicons. Possible correlations between phenotypic changes and genotypic variations will be examined. In addition an experimental plasmid phlogeny will be constructed that has the same topology as described in Project 1 (Experimental Evolution of Viruses), and which will permit us to test the ability of currently available algorithms and those developed in Project 4 to accurately reconstruct the phylogeny of a BHR plasmid that evolves in more than one genetic background.